Protein Purification

Abstract

The present invention provides methods for purifying proteins. In particular, the methods employ a two-step non-affinity ion exchange chromatography process without the use of an in-process tangential flow filtration step.

1 . A method for purifying a mixture, which comprises a target protein and one or more contaminants, comprising (a) subjecting the mixture to a cation exchange chromatography step followed by an anion exchange chromatography step and (b) isolating the target protein. 
   
   
       2 . A method for purifying a mixture, which comprises a target protein and one or more contaminants, which mixture has been partially purified using cation exchange chromatography, comprising (a) subjecting the mixture to an anion exchange chromatography step and (b) isolating the target protein. 
   
   
       3 . A method for purifying a mixture, which comprises an antibody and one or more contaminants, comprising (a) subjecting the mixture to cation exchange column chromatography followed by anion exchange chromatography and (b) isolating the target protein. 
   
   
       4 . The method of  claim 1 , wherein the contaminants are selected from host cell proteins, host cell metabolites, host cell constitutive proteins, nucleic acids, endotoxins, product related contaminants, lipids, media additives and media derivatives. 
   
   
       5 . The method of  claim 4 , wherein the target protein is purified to a purity of about 100 parts per million (ppm) or less of host cell proteins and about 10 pg/mg or less of nucleic acids. 
   
   
       6 . The method of  claim 1 , which further includes one or more additional steps selected from virus inactivation and virus removal. 
   
   
       7 . The method of  claim 1  wherein the anion exchange chromatography is performed at a pH of from about 4 to about 10 and at a conductivity of from about 0.1 to about 10 mS/cm. 
   
   
       8 . The method of  claim 1  wherein the anion exchange chromatography is performed at a pH of from about 6.0 to about 9.0 and at a conductivity of from about 0.5 to about 5 mS/cm. 
   
   
       9 . The method of  claim 1  wherein the anion exchange chromatography employs a membrane. 
   
   
       10 . The method of  claim 1  wherein the cation exchange chromatography is performed at a pH of from about 3 to about 10 and at a conductivity range of from about 0.1 to about 40 mS/cm. 
   
   
       11 . The method of  claim 1  wherein the cation exchange chromatography is performed at a pH of from about 4.0 to about 9.0 and at a conductivity of from about 0.5 to about 15 mS/cm. 
   
   
       12 . The method of  claim 1  wherein the cation exchange chromatography step employs a ligand selected from sulfonate, carboxylic, carboxymethyl sulfonic acid, sulfoisobutyl, sulfoethyl, carboxyl, sulphopropyl, sulphonyl, sulphoxyethyl and orthophosphate. 
   
   
       13 . The method of  claim 1  wherein the cation exchange chromatography step is performed on a resin or a membrane selected from Poros HS, Poros S, carboxy-methyl-cellulose, Sartobind S, BAKERBOND ABX™, sulphopropyl immobilized on agarose and sulphonyl immobilized on agarose, MonoS, MiniS, Source 15S, 30S, Capto S, SP sepharose, CM Sepharose, BAKERBOND Carboxy-Sulfon, WP CBX, WP Sulfonic, Hydrocell CM, Hydrocel SP, UNOsphere S, Macro-Prep High S, Macro-Prep CM, Ceramic HyperD S, Ceramic HyperD CM, Ceramic HyperD Z, Trisacryl M CM, Trisacryl LS CM, Trisacryl M SP, Trisacryl LS SP, Spherodex LS SP, DOWEX Fine Mesh Strong Acid Cation Resin, DOWEX MAC-3, Matrex Cellufine C500, Matrex Cellufine C200, Fractogel EMD SO3-, Fractogel EMD SE, Fractogel EMD COO—, Amberlite Weak and Strong Cation Exchangers, Diaion Weak and Strong Cation Exchangers, TSK Gel SP-5PW-HR, TSK Gel SP-5PW, Toyopearl CM (650S, 650M, 650C), Toyopearl SP (650S, 650M, 650C), CM (23, 32, 52), SE(52, 53), P11, Express-Ion C and Express-Ion S. 
   
   
       14 . The method of  claim 1  wherein the anion exchange chromatography step employs a ligand selected from quaternary ammonium or amine, dethylamine, diethylaminopropyl, amino, timethylammoniumethyl, trimethylbenzyl ammonium, dimethylethanolbenzyl ammonium, polyamine. 
   
   
       15 . The method of  claim 1  wherein the anion exchange chromatography step is performed using a resin or membrane selected from DEAE cellulose, Sartobind Q, MonoQ, MiniQ, Source 15Q, Source 30Q, ANX Sepharose Fast Flow, Q Sepharose high Performance, Capto Q, QAE SEPHADEX™, FAST Q SEPHAROSE™, WP PEI, WP DEAM, WP QUAT, Hydrocell DEAE, Hydrocell QA, UNOsphere Q, Macro-Prep DEAE, Macro-Prep High Q, Ceramic HyperD Q, ceramic HyperD DEAE, Spherodex LS DEAE, QMA Spherosil LS, QMA Spherosil M, Mustang Q, DOWEX Fine Mesh Strong Base Type I Anion Resins, DOWEX Fine Mesh Strong Base Type II Anion Resins, DOWEX MONOSPHER E 77, weak base anion from Dow Liquid Separations, Intercept Q membrane, Matrex Cellufine Q500, Matrex Cellufine A500 and Matrex Cellufine Q800, Fractogel EMD TMAE, Fractogel EMD DEAE and Fractogel EMD DMAE, and Amberlite. 
   
   
       16 . The method of  claim 1 , wherein the target protein is an antibody or antibody fragment. 
   
   
       17 . The method of  claim 16  wherein the antibody is a monoclonal antibody. 
   
   
       18 . The method of  claim 16  wherein the antibody is a fully human antibody. 
   
   
       19 . The method of  claim 16  wherein the antibody, or fragment thereof, is selected from single-chain antibody, diabody, linear antibody, bispecific antibody, multispecific antibody, defucosylated antibody, peptibody, Fab, Fab′, F(ab′) 2  and Fv. 
   
   
       20 . The method of  claim 1  wherein the target protein is an immunoadhesin. 
   
   
       21 . A method for manufacturing a protein of interest comprising culturing a host cell engineered to expresses the target protein, recovering the target protein in a mixture and purifying the mixture according to any one of  claim 1 . 
   
   
       22 . The method of  claim 21  wherein the protein of interest is an antibody or antibody fragment. 
   
   
       23 . The method of  claim 21 , further comprising the step of formulating the purified protein into a pharmaceutical composition.